Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth
Reexamination Certificate
2000-03-08
2003-12-30
Mehta, Ashwin D. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters plant part growth
C435S320100, C435S419000, C435S468000, C435S471000, C536S023600, C800S298000
Reexamination Certificate
active
06670526
ABSTRACT:
This is a 371 of PCT/GB98/01714, filed Jun. 12, 1998, which claims priority from GB 9712415.0, filed Jun. 13, 1997.
This invention relates to the genetic control of flowering in plants and the cloning and expression of genes involved therein. More particularly, the invention relates to the cloning and expression of the EARLY SHORT DAYS 4 gene of
Arabidopsis thaliana
, and homologues from other species, and manipulation and use of these genes in plants.
Efficient flowering in plants is important, particularly when the intended product is the flower or the seed produced therefrom. One aspect of this is the timing of flowering: advancing or retarding the onset of flowering can be useful to farmers and seed producers. An understanding of the genetic mechanisms which influence flowering provides a means for altering the flowering characteristics of the target plant. Species for which flowering is important to crop production are numerous, essentially all crops which are grown from seed, with important examples being the cereals, rice and maize, probably the most agronomically important in warmer climatic zones, and wheat, barley, oats and rye in more temperate climates. Important seed products are oil seed rape and canola, sugar beet, maize, sunflower, soybean and sorghum. Many crops which are harvested for their roots or leaves are, of course, grown annually from seed and the production of seed of any kind is very dependent upon the ability of the plant to flower, to be pollinated and to set seed. Delaying flowering is important in increasing the yield of plants from which the roots or leaves are harvested. In horticulture, control of the timing of flowering is important. Horticultural plants whose flowering may be controlled include lettuce, endive, spinach and vegetable brassicas including cabbage, broccoli and cauliflower, and carnations and geraniums.
Arabidopsis thaliana
is a facultative long day plant, flowering early under long days and late under short days. Because it has a small, well-characterized genome, is relatively easily transformed and regenerated and has a rapid growing cycle, Arabidopsis is an ideal model plant in which to study flowering and its control.
BRIEF DESCRIPTION OF THE INVENTION
The present inventors have discovered that one of the genes that regulates flowering time in Arabidopsis is a gene termed the EARLY SHORT DAYS 4 or ESD4 gene. The present inventors have found that plants carrying a recessive mutation affecting the ESD4 gene flower earlier than their wild-types under long and short days. The ESD4 gene has now been cloned and sequenced and the inventors have demonstrated that the mutation is a deletion removing part of the gene. This provides indication that reducing or abolishing ESD4 function accelerates flowering, and therefore that the ESD4 gene likely encodes a repressor of flowering.
DETAILED DESCRIPTION OF THE INVENTION
According to a first aspect of the present invention there is provided a nucleic acid molecule including a nucleotide sequence encoding a polypentide with ESD4 function. Those skilled in the art will appreciate that “ESD4 function” may be used to refer to the ability to influence the timing, of flowering phenotypically when its expression is reduced like the ESD4 gene of
Arabidopsis thaliana
. esd4 mutants exhibit early flowering under long and short days, the timing of flowering being substantially unaffected by vernalisation.
The present invention provides a nucleic acid isolate encoding a polypeptide including the amino acid sequence shown in
FIG. 1
, which may include the coding sequence shown in
FIG. 1
which is that of the ESD4 gene of
Arabidopsis thaliana
.
FIG. 2
shows a genomic sequence including nucleotides encoding the polypeptide for which the amino acid sequence is shown in FIG.
1
.
Nucleic acid according to the present invention may have the sequence of an ESD4 gene of
Arabidopsis thaliana
, or be a mutant, variant, derivative or allele or a homologue of the sequence provided. Preferred mutants, variants, derivatives and alleles are those which encode a protein which retains a functional characteristic of the protein encoded by the wild-type gene, especially the ability to affect a physical characteristic of a plant, such as a flowering characteristic, especially the ability to repress flowering as discussed herein.
A mutant, variant, derivative or allele in accordance with the present invention may have the ability to affect a physical characteristic of a plant, particularly a flowering characteristic. In various embodiments a mutant, variant, derivative or allele represses flowering compared with wild-type on expression in a plant, e.g. compared with the effect obtained using a gene sequence encoding the polypeptide of FIG.
1
. “Repression” of flowering delays, retards, inhibits or slows it down. In other embodiments, a mutant, variant, derivative or allele promotes flowering compared with wild-type on expression in a plant, e.g. compared with the effect obtained using a gene sequence encoding the polypeptide of FIG.
1
. “Promotion” of flowering advances, accelerates or brings it forward. Comparison of effect on flowering or other characteristic may be performed in
Arabidopsis thaliana
, although nucleic acid according to the present invention may be used in the production of a wide variety of plants and for influencing a characteristic thereof.
As discussed further below, over-expression of nucleic acid according to the present invention may delay flowering while under expression may promote flowering in a transgenic plant.
Changes to a sequence, to produce a mutant, variant or derivative, may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide. Of course, changes to the nucleic acid which make no difference to the encoded amino acid sequence are included.
A preferred nucleic acid sequence for an ESD4 gene including a coding sequence according to the present invention is shown in
FIG. 1
, along with the predicted amino acid sequence of a polypeptide according to the present invention which has ESD4 function.
A mutant, allele, variant or derivative amino acid sequence in accordance with the present invention may include within the sequence shown in
FIG. 1
, a single amino acid change with respect to the sequence shown in
FIG. 1
, or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80 or 90 changes. In addition to one or more changes within the amino acid sequence shown in
FIG. 1
, a mutant, allele, variant or derivative amino acid sequence may include additional amino acids at the C-terminus and/or N-terminus.
A sequence related to a sequence specifically disclosed herein shares homology with that sequence. Homology may be at the nucleotide sequence and/or amino acid sequence level. Preferably, the nucleic acid and/or amino acid sequence shares homology with the coding sequence or the sequence encoded by the nucleotide sequence of
FIG. 1
, preferably at least about 50%, or 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology.
As is well-understood, homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for “conservative variation”, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. Similarity may be as defined and determined by the TBLASTN program, of Altschul et al. (1990) J. Mol. Biol. 215: 403-10, which is in standard use in the art, or, and this may be preferred, the standard program BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711). BestFit mak
Mehta Ashwin D.
Nixon & Vanderhye P.C.
Plant Bioscience Limited
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